Illumina-Based RNA Sequencing Protocol

RNA sequencing was performed by the Washington University Genome Technology Access Center) as we reported^{51 (link)}. Library preparation was performed with 10 μG of total RNA with a Bioanalyzer RIN score >8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was fragmented and reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies) and random hexamers. A second strand reaction was performed to yield ds-cDNA, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were amplified for 12 cycles and sequenced on an Illumina HiSeq-3000 using single reads extending 50 bases. RNA-seq reads were aligned to the Ensembl release 76 top-level assembly with STAR version 2.0.4b. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.5. Transcript counts were produced by Sailfish version 0.6.3.